Actionability classification of variants of unknown significance correlates with functional effect

Genomically-informed therapy requires consideration of the functional impact of genomic alterations on protein expression and/or function. However, a substantial number of variants are of unknown significance (VUS). The MD Anderson Precision Oncology Decision Support (PODS) team developed an actionability classification scheme that categorizes VUS as either “Unknown” or “Potentially” actionable based on their location within functional domains and/or proximity to known oncogenic variants. We then compared PODS VUS actionability classification with results from a functional genomics platform consisting of mutant generation and cell viability assays. 106 (24%) of 438 VUS in 20 actionable genes were classified as oncogenic in functional assays. Variants categorized by PODS as Potentially actionable (N = 204) were more likely to be oncogenic than those categorized as Unknown (N = 230) (37% vs 13%, p = 4.08e-09). Our results demonstrate that rule-based actionability classification of VUS can identify patients more likely to have actionable variants for consideration with genomically-matched therapy.


NTRK3 G623R
The functional effect of this alteration on NTRK3 kinase activity has not been experimentally determined. It is located within the kinase domain of the protein (amino acids 538-839, UniProt 7 ). Gain-of-function missense mutations have been reported in this region 8 . However, we are currently unaware of any experimental data demonstrating an increase in kinase activity with this specific mutation. NTRK3 G623R confers resistance to FDA approved Trk inhibitors larotrectinib and entrectinib. NTRK3 623R is referred to as a solvent front mutation that confers resistance by sterically hindering drug binding [9][10][11] . This alteration is not reported in dbSNP or ClinVar 6 databases (June, 2022).

Unknown
Yes:Literature based (for resistance to larotrectinib or entrectinib within the context of a susceptible NTRK3 fusion)

AKT1 D323E
This alteration is considered activating. This alteration is located within the kinase domain (amino acids 150-408, UniProt 7 ), which is essential for the function of AKT1 12,13 . Expression of AKT1 D323E in BaF3 and MCF10A cells resulted in moderately increased cell viability and/or growth compared with expression of the wildtype gene in the Institute of Personalized Cancer Therapeutics Functional Genomics laboratory (Unpublished Observations). There is currently no published data detailing the functional consequence of this alteration. This alteration is not reported in dbSNP or ClinVar 6 (June, 2022) databases.

Activating
Yes: Functional Genomics (for treatment with AKT or mTOR inhibitors)

BRCA1 A1708E
This alteration has been reported to be inactivating. A1708E is also called c.5123C>A and is reported to be a founder BRCA1 mutation in Hispanic populations of breast and/or ovarian cancer 14,15 . Causality analysis, which takes into consideration that this variant segregates primarily with diseased populations and rarely co-occurs with other deleterious BRCA1 variants, shows A1708E favors causality for cancer risk [16][17][18] . This pathogenicity is confirmed by multiple functional studies that show A1708E strongly disrupts BRCA1 stability and activity [19][20][21][22][23][24][25][26][27] . Furthermore, c.5123C>A has been shown to alter BRCA1 exon 18 splicing showing partial in-frame skipping of exon 18, which encodes a portion of the BRCT domain, which is either the primary mechanism for A1708E's loss-of-function effect or it contributes to BRCA1 inactivation 28 . A genomic alteration resulting in this amino acid change is recorded within dbSNP (rs28897696  29,30 . This alteration is not reported in dbSNP or ClinVar 6 (June, 2022) databases.

Activating: Inferred
Yes: Inferred (for treatment with gamma secretase inhibitors)

BRCA1 K223fs*11
Although this alteration has not been experimentally tested, it is inferred to be inactivating. This alteration has not been reported in dbSNP or ClinVar 6 (June, 2022) databases. This alteration results in a shift in the coding frame of BRCA1 at codon 223, and premature protein truncation 11 amino acids downstream. K223fs*11 introduces a truncation within the region that interacts with the SWI/SNF chromatin remodeling complex subunit BRD7 (amino acids 1-300 31 ), which facilitates the recruitment of BRCA1 to the promoter of specific genes, such as ER alpha . This event eliminates translation of the vast majority of the 1863 amino acid protein, including the essential BRCT1/2 (1642-1736, 1756-1855) and SCD (1280-1524) domains and the regions that interact with FANCA, Rad51 (740-1083), and PALB2 (1397-1424) (UniProt 7 ). BRCT1/2 domains interact with various cell cycle checkpoints, repair proteins, chromatin remodeling, and transcription factors 32,33 ; SCD is important for BRCA1-mediated checkpoint activation; PALB2-BRCA1 interaction serves as the molecular scaffold 33,34 , which are all essential to the tumor suppressor function of BRCA1. Collectively, this alteration is predicted to result in loss-offunction due to disruption and/or elimination of essential protein domains.

Inactivating: Inferred
Yes: Inferred (for treatment with PARP inhibitors and platinum-based chemotherapy)

ERBB2 G727A
This alteration has not been functionally characterized. G727A alteration occurs within the kinase domain (amino acids 720-987) of ERBB2 (UniProt 7 ). Mutations within this region of ERBB2 have been previously reported in tumors and are highly activating mutations [35][36][37] . Thus, there is the potential that this alteration confers a similar effect; however, it has not been experimentally determined. This alteration is not reported in dbSNP or ClinVar 6 databases (June, 2022).

Unknown
Potentially (for treatment with HER2 inhibitors)

ERBB2 Q646H
This alteration is of unknown functional significance. It is not located within a functionally characterized domain (UniProt 7 ). This alteration is not reported in dbSNP or ClinVar 6 databases (May, 2022).

Unknown
Unknown (for treatment with HER2 inhibitors)

BRCA1 V191I
This alteration was characterized to result in neutral effect on BRCA1 function. V191I is located close to the ring finger domain 38 . Functional studies demonstrated that this alteration resulted in comparable function to the wild type in homologous directed recombination, proliferation, and cisplatin sensitivity assays 25,39 . A genomic alteration resulting in this amino acid change is recorded within dbSNP (rs80357090  40 . An in vitro model suggested that the lack of kinase domain interrupted ERBB2 dimerization and activation process 41 . Therefore, this alteration is predicted to be inactivating. This alteration is not reported in dbSNP or ClinVar 6 databases (June, 2022).

Inactivating: Inferred
No (not actionable for treatment with HER2 inhibitors)

AKT1 A58V
This alteration is of likely benign functional significance. Expression of this alteration in BaF3 and MCF10A cells resulted in no change in cell viability and/or growth compared to the wildtype in the IPCT Functional Genomics laboratory. A58V occurs at a conserved residue and is located within the N-terminal pleckstrin homology (PH) domain 7,42 , which recruits AKT1 to the plasma membrane and is required for AKT1 activation 43 . A genomic alteration leading to this amino acid change is recorded in dbSNP (rs1892948595, May, 2022). It is not reported within ClinVar 6 (June, 2022).

Likely Benign
No: Functional Genomics (not actionable for treatment with AKT or mTOR inhibitors) Supplementary   80 . Expression of CBL Y371N within BaF3 cells led to an increase in cellular proliferation and/or viability within the IPCT Functional Genomics laboratory when compared with expression of the wildtype gene. CBL is typically considered a tumor suppressor 81 ; however, expression of the wildtype CBL gene had no effect when compared with expression of a negative control. Thus, it is unclear by what mechanism expression of the mutation led to increased cellular proliferation and/or viability. One hypothesis is that the mutant serves as a dominant negative inhibiting the function of the endogenous CBL protein, but this has not been experimentally tested. While the functional effect of this mutation has not been tested within the published literature, Y371F was investigated. This mutation impaired CBL-mediated EGFR ubiquitination and degradation and diminished the loss of phosphorylation seen with mutation of Y700 and Y774 82 .  . Other alterations at the same codon are known to be inactivating, R173C [83][84][85] and R173H 83,86 . However, expression of this variant in BaF3 and MCF10A cells resulted in no change in cell viability and/or growth compared with expression of the wildtype gene in the IPCT Functional Genomics laboratory. Given that other variants at this codon confer a loss-of-function and the mutation is classified as likely pathogenic in ClinVar 6 (VCV000428258.5), it is possible that this alteration does effect phosphatase activity but does not confer a change in cell survival in this specific assay.  86 . Other cancer-and disease-associated mutations located within this region, have been shown to be inactivating and tumor promoting mutations [87][88][89] . In fact, another alteration of the same codon, T167A, was experimentally characterized to decreases PTEN phosphatase activity 86 . However, expression of this variant in BaF3 and MCF10A cells resulted in no change in cell viability and/or growth compared with expression of the wildtype gene in the IPCT Functional Genomics laboratory. T167I has been previously reported in endometrial cancer 90 . Given that other variants at the same codon confer a loss-of-function, it is possible that this alteration does effect phosphatase activity but does not confer a change in cell survival in this specific assay.

STK11 P314H
Activating Activating Activating Unknown Potentially This mutation is of unknown functional significance within the published literature. It has been reported in a patient with Peutz-Jeghers syndrome, a rare autosomal-dominant disorder characterized by the formation of benign hamartomatous polyps in the gastrointestinal tract 91 . Germline STK11 mutations are found in approximately 70% of patients with near complete penetrance in patients harboring mutations in the gene 91 . This alteration was also detected as a somatic mutation in a patient with colorectal cancer 92 . P314 is located just C-terminal to the kinase domain (amino acids 49-309, UniProt 7 ), but not within a functionally characterized region. Expression of STK11 P314H in BaF3 and MCF10A cells resulted in a weak increase in cell viability and/or growth compared with expression of the wildtype gene in the IPCT Functional Genomics laboratory. STK11 is generally considered a tumor suppressor gene 93 ; however, expression of the wildtype gene had no effect on cellular proliferation or survival. Thus, it is unclear by what mechanism expression of the mutation led to increased cellular proliferation and/or viability. There is currently no published data detailing the functional consequence of this alteration. The clinical significance of this alteration in ClinVar 6 is uncertain significance (VCV001327848.1; Jan, 2022).